MYBA and MYBPA transcription factors co-regulate anthocyanin biosynthesis in blue-coloured berries.

The regulatory network of R2R3 MYB transcription factors in anthocyanin biosynthesis is not fully understood in blue-coloured berries containing delphinidin compounds. We used blue berries of bilberry (Vaccinium myrtillus) to comprehensively characterize flavonoid-regulating R2R3 MYBs, which revealed a new type of co-regulation in anthocyanin biosynthesis between members of MYBA-, MYBPA1- and MYBPA2-subgroups. VmMYBA1, VmMYBPA1.1, and VmMYBPA2.2 expression was elevated at berry ripening and by abscisic acid treatment. Additionally, VmMYBA1 and VmMYBPA1.1 expression was strongly down-regulated in a white berry mutant. Complementation and transient overexpression assays confirmed VmMYBA1 and VmMYBA2 to induce anthocyanin accumulation. Promoter activation assays showed that VmMYBA1, VmMYBPA1.1, and VmMYBPA2.2 had similar activity towards DFR and ANS, but differential regulation activity for UFGT and F3'5'H promoters. Silencing of VmMYBPA1.1 in berries led to the down-regulation of key anthocyanin and delphinidin biosynthesis genes. Functional analyses of other MYBPA-regulators, and a member of novel MYBPA3-subgroup, associated them with proanthocyanidin biosynthesis and F3'5'H expression. The existence of 18 flavonoid-regulating MYBs indicated gene duplication, which may have enabled functional diversification among MYBA-, MYBPA1- and MYBPA2-subgroups. Our results provide new insights into the intricate regulation of the complex anthocyanin profile found in blue-coloured berries involving regulation of both cyanidin and delphinidin branches. This article is protected by copyright. All rights reserved.